Evaluation and optimization of content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia / 中国中药杂志

This study discovered that the resolution of 3,5-O-dicaffeoylquinic acid(isochlorogenic acid A) in the content determination method of Chrysanthemi Flos in Chinese Pharmacopoeia(ChP)(2020 edition) was poor, which affected accurate quantification. We tested the method in ChP with chromatographic columns of seven brands to clarify the problems in the existing method, optimized the chromatographic conditions by adjusting the mobile phase composition and elution ratio and replacing the chromatographic column packing, and carried out the reproducibility assay for the new method. The two methods were compared for the content determination results of Chrysanthemi Flos prepared from six different varieties. As evaluated by the resolution based on different chromatographic columns of seven brands, the existing method failed to separate isochlorogenic acid A and isochlorogenic acid D well. The peaks of the two components were not completely separated on three chromatographic columns, and isochlorogenic acid A and isochlorogenic acid D generated a co-effluent peak in the other four columns. Isochlorogenic acid A and isochlorogenic acid D could be completely separated under the optimized chromatographic conditions. The difference in the peak areas of isochlorogenic acid A+isochlorogenic acid D obtained by the optimized method and the method in ChP was not significant, with deviation less than 3.0%, which further proved that the result measured by the method in ChP was the co-effluent of isochlorogenic acid A and isochlorogenic acid D. The optimized method can ensure the accurate quantification of isochlorogenic acid A. The existing content determination method of Chrysanthemi Flos has the problem of poor resolution. It is recommended to revise the chromatographic conditions for the content determination method of Chrysanthemi Flos to improve the resolution of isochlorogenic acid A and ensure its accurate quantification.

Simultaneous Determination of Seven Constituents in Qinggan Lidan Mixture by HPLC / 中国实验方剂学杂志

Objective: To establish an HPLC method for simultaneous determination of geniposide,chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid A,B and C in Qinggan Lidan mixture,in order to provide references for its quality control. Method: The analysis of methanol extract of this drug was performed on a 35℃ Luna C18 column (4.6 mm×250 mm,5μm),with the mobile phase comprised of acetonitrile-0.4% phosphoric acid flowing at 1.0 mL·min-1 in a gradient elution mode (0-10 min,8%-12%A;10-30 min,12%A;30-60 min,12%-35%A),and the detection wavelengths were set at 238 and 327 nm. Result:Geniposide,chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid A,B and C were completely separated,and well separated from other constituents. The linear ranges of geniposide,chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid A,B and C were 0.188-2.355,0.083-1.040,0.074-0.920,0.075-0.940,0.064-0.800,0.076-0.955,0.071-0.888 μg (r ≥ 0.999 0),respectively. The average recoveries were 99.45%,98.45%,99.06%,98.50%,98.16%,101.01%,96.93%,with the RSDs of 0.5%,1.8%,1.3%,2.4%,2.3%,1.6%,1.6%,respectively.The contents of geniposide,chlorogenic acid,neochlorogenic acid,cryptochlorogenic acid,isochlorogenic acid A,B and C were 3.420-3.794,0.835-0.890,1.222-1.275,1.064-1.210,0.377-0.398,0.419-0.464 and 0.362-0.405 g·L-1,respectively. Conclusion:This method can be used for simultaneous determination of muti-ingredients in Qinggan Lidan mixture,and the established method is simple and accurate,with a good reproducibility and high sensitivity. It can be used for the quality control of Qinggan Lidan mixture.

Isochlorogenic acid A inhibits activation of NLRP3/NF-ΚB in rats with collagen-induced arthritis / 中国药理学通报

Aim To analyse the anti-inflammatory effect of isochlorogenic acid A in rats with collagen-induced arthritis and the potential mechanism . Methods Male SD rats were randomly divided into normal group, model group, isochlorogenic acid A high and low dose groups. The model was constructed in all groups except control group. After continuous administration for six weeks, the plantar thickness and spleen organ coefficient were measured in rats. NLRP3, caspase-1, NF-ΚB p65, P-NF-ΚB p65, P-IΚB and RANKL protein in synovium of joint were detected by Western blot, and IL-lβ, IL-6, TNF-α, CRP, IFN-γ, IL-18 expressions in blood plasma were detected by ELISA kit. Results Compared with normal group, the swelling degree of plantar and the spleen organ coefficient in model group increased significandy. Western blot showed that the expression of NLRP3, caspase-1, NF-ΚB p65, P-NF-ΚB p65, P-IΚB-O and RANKL protein were up-regulated in synovium of joint tissues in model group. ELISA showed that the expression of blood plasma IL-lβ, IL-6, TNF-α, CRP, IFN-γ and IL-18 were significantly up-regulated in model group. Different doses of isochlorogenic acid A can significantly reduce the performance of the above indicators in model group. Conclusions Isochlorogenic acid A has a good anti-inflammatory effect on collagen-induced arthritis, and its anti-inflammatory activity may be related to the decrease of NLRP3 inflammatory complex activation and NF-ΚB phosphorylation.

Simultaneous determination of 12 components in Qiju Dihuang Oral Liquid by HPLC / 中草药

Objective: To establish a rapid, accurate, and practical HPLC method for simultaneous determination the content in Qiju Dihuang Oral Liquid (QDOL) of 5-HMF, morroniside, chlorogenic acid, cryptochlorogenic acid, loganin, paeoniflorin, verbascoside, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and paeonol. Methods: YMC ODS column (250 mm × 4.6 mm, 5 μm) was used, column temperature was set at 35 ℃, gradient elution with 0.1% formic acid aqueous solution- acetonitrile was used as mobile phase, flow rate was 1.0 mL/min, detection wavelength was 254 and 325 nm. The injection volume was 10 μL. Results: The injection amount of 5-HMF, morroniside, chlorogenic acid, cryptochlorogenic acid, loganin, paeoniflorin, verbascoside, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, paeonol injection quality at 0.08—1.60, 0.12—2.40, 0.09—1.80, 0.06—1.20, 0.10—2.00, 0.30—6.00, 0.01—0.20, 0.01—0.20, 0.01—0.20, 0.005—0.10, 0.005—0.10, and 0.01—0.20 μg showed a good linear relationship with peak area, with good precision, repeatability and stability. The recovery rates of the samples were between 96% and 103%, the RSD was 2.13%, 3.45%, 2.86%, 2.59%, 3.15%, 3.49%, 2.19%, 3.25%, 2.37%, 2.53%, 2.91%, and 3.35%, respectively. The content of each component of the five batches of samples was stable, and the mass concentrations range of the 12 components tested were 98.56—102.56, 204.28—212.10, 18.53—18.89, 1.95—2.05, 12.31—12.54, 87.01—87.12, 5.35—5.43, 16.08—16.15, 8.69—8.72, 8.89—8.95, 5.12—5.19, and 1.87—1.94 μg/mL. Conclusion: The method simltaneosly determines the content of 5-HMF, morroniside, chlorogenic acid, cryptochlorogenic acid, loganin, paeoniflorin, verbascoside, luteoloside, isochlorogenic acid B, isochlorogenic acid A, isochlorogenic acid C, and paeonol in QDOL, which is suitable for the quality control of QDOL.

Rapid determination of seven components in Angelica sinensis based on near infrared spectroscopy / 中草药

Objective: To established a rapid nondestructive determination method for the multi-marker constituents in Angelica sinensis by near infrared spectroscopy (NIRS) combined with the partial least squares (PLS) method in order to improve the quality control for A. sinensis. Methods: A total of 108 batches of A. sinensis from different origins were collected for the research. An Ultra performance liquid chromatography (UPLC) method was established to measure the content of the seven components in A. sinensis, which were adopted as the reference value. And the integrating sphere diffuse reflection mode was employed to collect the NIR spectrum. The quantitative calibration model between the near infrared spectrum and the reference content of each component to be measured was established by PLS and other chemometrics methods. Each part of the modeling process was optimized respectively, including the selection of calibration set and validation set, different pretreatment methods and different spectral section. Results: The correlation coefficient for calibration set of chlorogenic acid, ferulic acid, isochlorogenic acid A, ligustilide, butylidenephthalide, senkyunolide I and levistilide A were 0.937 6, 0.970 2, 0.963 4, 0.991 1, 0.962 4, 0.966 6 and 0.947 6, respectively; The root mean square error of prediction (RMSEP) were 0.072 1, 0.038 9, 0.011 3, 0.483 0, 0.017 5, 0.178 0 and 0.097 0, respectively. The predicted values of NIRS models and the measured values of UPLC showed a good linear relation, which presented a great prediction ability of the models. Conclusion: The methods of NIRS combined with PLS can be applied for the rapid content determination of seven components in A. sinensis including chlorogenic acid, ferulic acid, isochlorogenic acid A, ligustilide, butylidenephthalide, senkyunolide I and levistilide A, which is proved to be simple and reliable.

Simultaneous determination of ten components in Lonicerae Japonicae Flos from different habitats by UPLC / 中草药

Objective: To establish a method for the determination of seven organic acids, one flavone and two iridoid terpenoids in Lonicerae Japonicae Flos from different habitats. Methods: The content of 10 components in Lonicerae Japonicae Flos from different habitats were determined by ultra-performance liquid chromatography (UPLC). The mobile phase for gradient elution was 0.1% phosphoric acid solution (A)- methanol (B); The flow rate was 0.3 mL/min, and the column temperature was 30 ℃. Results: A UPLC method for simultaneous determination of seven organic acids, one flavone and two iridoid terpenoids in Lonicerae Japonicae Flos was established. Principal component analysis (PCA) and partial least squares method (PLS-DA) were used to analyze the distribution and characteristics of 10 constituents of Lonicerae Japonicae Flos collected from different habitats; Three production areas of Shandong, Jiangsu and Shaanxi are respectively grouped into one group. Conclusion: The method is stable and feasible, which could be used as a reference for evaluating the quality of Lonicerae Japonicae Flos in a more comprehensive way.

Simultaneous determination of Forsythoside A and other four components in Xiao'er-Ganmao-Keli from multivendor by HPLC / 国际中医中药杂志

Objective To establish a method for the simultaneous determination of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A by HPLC, and test 16 batches of samples from 14 manufacturers. Methods The test was performed on Kinetex EVO C18 column (150 mm × 4.6 mm, 5 μm) with the column temperature at 35 ℃ . The gradient elution was adopted with the mobile phase of acetonitrile and 0.3% phosphoric acid aqucous at a flow rate of 1.0 ml/min. The detection wavelength of (R,S)-Epigoitrin and Phillyrin were set as 236 nm, the detection wavelength of Forsythoside A, Cholorogenic Acid and Isochlorogenic Acid A were set as 327 nm. Results The good linear relationships were displayed within the linear range of 0.050 45-2.018 00 μg for Forsythoside A (r=0.999 9), 0.018 21-0.728 40 μg for Phillyrin (r=0.999 9), 0.010 16-0.406 40 μg for (R,S)-Epigoitrin (r=0.999 9), 0.006 60-0.263 90 μg for Cholorogenic Acid (r=0.999 9) and 0.0040 44~0.161 76 μg for Isochlorogenic Acid A ( r=0.999 5). The RSDs of reproducibility and stability tests were lower than 2%; recoveries were 97.01%, 98.28%, 99.35% and 96.21%, RSD were 3.19%, 1.19%, 0.81%, 2.88% and 2.96%. The content ranges of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A from 16 batches of samples from 14 manufacturers were 0.057 43-1.508 71 mg/g, 0.017 72-0.350 15 mg/g, 0.005 68-0.177 13 mg/g, 0.007 53-0.226 33 mg/g and 0.00308-0.11908 mg/g. Conclusions The established method is simple and accurate, and has a good repeatability. It can be used for the quality analysis of Forsythoside A, Phillyrin, (R,S)-Epigoitrin, Chlorgenic Acid and Isochlorogenic Acid A. The content of the tested chemical components from 16 batches of samples from 14 manufacturers have significant differences which indicate that a reinforcement of the quality control is needed.

Determination of Isochlorogenic Acid A and Isochlorogenic Acid C in Vernonia Anthelmintica by High Performance Liquid Chromatography / 医药导报

Objective To establish the chromatographic conditions of isochlorogenic acid A and isochlorogenic acid C in vernonia anthelmintica. Methods By changing the mobile phase,flow rate,column temperature and other chromatographic conditions,the best chromatographic conditions was we pursued to established. Results The linear relationship between the concentration of isochlorogenic acid A and the peak area was between 5. 825-69. 9 μg·mL-1, and the concentration of isochlorogenic acid C,was between 5.15-61.80 μg·mL-1and the peak area was good . The sample recovery rates of the two groups were 98.70%-101.92%(RSD＝1.04%,n＝9)、95.99%-102.52%(RSD＝1.90%,n＝9). Conclusion The method is simple,rapid, accurate and reliable for the determination of isochlorogenic acid A and isochlorogenic acid C in Vernonia anthelmintica and also for the quality control of the raw material.

Simultaneous determination of 10 components in Yinhuang preparations by UPLC / 中草药

Objective: To develop an UPLC method for simultaneous determination the contents of 10 active constituents [neochlorogenic acid (NCA), chlorogenic acid (CA), cryptochlorogenic acid (CCA), isochlorogenic acid A (ICAA), isochlorogenic acid B (ICAB), isochlorogenic acid C (ICAC); bacicalin, wogonoside, baicalein, and wogonin] in Yinhuang preparations. Methods: Analysis was performed on an Acquiyt UPLC BEH C18 chromatographic column (100 mm × 2.1 mm, 1.7 μm) eluted with acetonitrile- 0.4% phosphoric acid, gradient elution, and the flow rate was 0.4 mL/min, the detection wavelength was set at 326 nm, the column temperature was 40℃, and the injection volume was 1.0 μL. Results: All calibration curves were linear (r ≥ 0.999 6) over the tested ranges. The average recoveries (n = 9) ranged from 97.43% to 99.94% with RSD value below 2.0%. The contents of 11 batches of NCA, CA, CCA, ICAA, ICAB, ICAC, bacicalin, wogonoside, baicalein, and wogonin were 0.236-3.419, 5.279-26.220, 0.495-4.714, 0.130-2.702, 0.310-3.210, 0.363-5.036, 35.209-133.289, 1.493-6.635, 1.546-5.393, and 0.254-0.823 mg/g. Conclusion: This method is rapid, simple, sensitive, accurate, and reproducible, which can be used for quality control of Yinhuang preparation, and provide a reference for the formulation of quality standards to enhance in the future.

Simultaneous Determination of 4 Components in Xiaoer Ganmao Granule by HPLC / 中国药房

OBJECTIVE:To establish a method for the simultaneous determination of(R,S)-goitrin,chlorogenic acid,luteolo-side and isochlorogenic acid A in Xiaoer ganmao granule. METHODS:HPLC was performed on the column of Hedera C18 with mo-bile phase of acetonitrile-0.1% formic acid aqueous at a flow rate of 1.0 ml/min,the detection wavelength was 254 nm,330 nm and 370 nm,column temperature was 40 ℃,and the injection volume was 5 μl,RESULTS:The linear range was 6.6-105 μg/ml for (R,S)-goitrin (r=0.999 9),9-140 μg/ml for chlorogenic acid (r=0.999 9),9-144 μg/ml for luteoloside (r=0.999 8) and 9-138 μg/ml for isochlorogenic acid A(r=0.999 6);the limits of quantitation were 330 ng,450 ng,450 ng and 450 ng,limits of detection were 66 ng,90 ng,90 ng and 90 ng,respectively;RSDs of precision,stability and reproducibility tests were lower than 3%;recoveries were 95.01%-98.77%(RSD=1.48%,n=6),95.14%-98.91%(RSD=1.52%,n=6),95.11%-97.54%(RSD=0.93%,n=6) and 95.58%-99.63%(RSD=1.73%,n=6). CONCLUSIONS:The method is simple and accurate,and suitable for the simultaneous determination of(R,S)-goitrin,chlorogenic acid,luteoloside and isochlorogenic acid A in Xiaoer ganmao granule.

Isochlorogenic acid A affects P450 and UGT enzymes in vitro and in vivo / 中国天然药物

Isochlorogenic acid A (ICQA), which has anti-inflammatory, hepatoprotective, and antiviral properties, is commonly presented in fruits, vegetables, coffee, plant-based food products, and herbal medicines. These herbal medicines are usually used in combination with other medicines in the clinic. However, little is known about the regulatory effects of ICQA on drug-metabolizing enzymes and the herb-drug interactions. In the present study, we evaluated the inhibitory potentials of ICQA on CYP1A2, CYP2C9, CYP2C19, CYP3A4, CYP2D6, and CYP2E1 in vitro based on a cocktail approach. The P450 and UGT activities in mice treated with ICQA for a prolonged period were also determined. Our results demonstrated that ICQA exhibited a weak inhibitory effect on CYP2C9 in human liver microsomes with IC being 57.25 μmol·L and Ki being 26.77 μmol·L. In addition, ICQA inhibited UGT1A6 activity by 25%, in the mice treated with ICQA (i.p.) at 30 mg·kg for 14 d, compared with the control group. Moreover, ICQA showed no mechanism-based inhibition on CYP2C9 or UGT1A6. In conclusion, our results further confirm a safe use of ICQA in clinical practice.

Simultaneous determination of chlorogenic acid, aesculetin, isochlorogenic acid B, and isochlorogenic acid A in Cichorii Herba by QAMS / 中草药

Objective: To establish the quantitative analysis of multi-components by single marker (QAMS) method to determine the content of chlorogenic acid, aesculetin, isochlorogenic acid B, and isochlorogenic acid A in Cichorii Herba (chicory). Methods: Selecting aesculetin as the internal reference substance, the relative correction factors (RCF) of chlorogenic acid, isochlorogenic acid B and isochlorogenic acid A were established respectively, and then the contents of the three constituents were calculated by RCF, to achieve the quality of chicory through QAMS. At the same time, the external standard method was used to determine the content of four constituents in chicory and compare the difference between calculated values and measured values, so as to verify the construction method for accuracy, applicability, and repeatability. Results: No significant difference was observed in chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A from eight batches of chicory in the quantitative results by these two methods. The validated QAMS method had good precision, reproducibility, and reliability. Conclusion: The established QAMS method is suitable and feasible for the quality control of chicory.

Analysis on chemical constituents in Bupleuri Radix by HPLC-Q-TOF-MS / 中草药

Objective: To investigate the chemical constituents in extract of Bupleuri Radix using HPLC-Q-TOF-MS. Methods: The HPLC separation achieved on Agilent 1200 HPLC Diamonsil II C18 column (250 mm×4.6 mm, 5 μm) was used with a mobile phase of 0.05% H2O-formic acid (A) and acetonitrile (B), the flow rate was 1.0 mL/min, the column temperature was 35℃, and the detection wavelength was 200-600 nm. Positive and negative ions MS information and element analysis, via comparing MS data with those of the standard compounds and coupled with the related literature were used to analyze the main chemical constituents of BupleuriRadix. Results: Twenty-one chemical constituents were identified, including three phenolic acid, four flavonoids, and fourteen triterpenoid saponins, and isochlorogenic acid A (5), isochlorogenic acid B (6), 7,3'-di-O-methylquercetin (8), and 5-hydroxy-7-acetoxyflavone (24) were separated from Bupleuri Radix for the first time. Conclusion: It is used to quickly analyze the chemical constituents from Bupleuri Radix by establishing a rapid accurate evaluation method using HPLC-Q-TOF-MS, which could provide the references for the application development.

Quality assessment of Shuangyu Granules based on simultaneous determination by HPLC fingerprint and multi-components / 中草药

Objective: To establish an HPLC fingerprint chromatography and determine seven compounds of multi-components in Shuangyu Granules (SG). Methods: The Kromasil C18 (250 mm × 4.6 mm, 3.5 μm) column was used with a mobile phase of acetonitrile-0.05% trifluoroacetic acid gradient elution. The flow rate was 0.8 mL/min, the column temperature was 30℃, and the detection wavelengths were 230 and 327 nm. The common peaks were identified by Q-TOF/MS. Results: The fingerprint chromatography included 17 mutual peaks, and the similarity was more than 0.95. Fourteen common peaks had been identified by LC-Q-TOF/MS, seven of which were unequivocally identified via comparing the retention times and mass spectra data with those of the standard compounds. Then the seven marker components were quantified. The developed quantitative method was validated in terms of accuracy (the recoveries ranged from 97.8% to 101.8% with RSDs less than 2%). Conclusion: The method is rapid, simple, and accurate and can be used for the quality control of SG.

Preparation technology of isochlorogenic acids A, B, and C / 中草药

Objective: To establish a method for separation of isochlorogenic acids A, B, and C from Lonicerae Flos. Methods: Isochlorogenic acids A, B, and C in Lonicerae Flos were isolated and purified by macroporous resin and medium-low-pressure preparative chromatography. Their structures were identified on the basis of the spectral data and physicochemical property. Results: The contents of prepared isochlorogenic acids A, B and C were 98.7%, 99.2%, and 97.6%, respectively. Conclusion: This method is economic, simple, rapid, and effective for the preparation of isochlorogenic acids A, B, and C with high purity.

Simultaneous determination of the contents and fingerprint of multi-components in honeysuckle extract by HPLC / 中国药学杂志

OBJECTIVE: To establish a HPLC method for the simultaneous determination of the contents and fingerprint of 7 active constituents (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, and isochlorogenic acid C) in honeysuckle extract. METHODS: The chromatographic separation was achieved on a AkzoNobel Kromasil C18 column(4.6 mm × 250 mm, 5 μm), the mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution with gradient elution at a flow rate of 1.0 mL · min-1, the detection wavelength was set at 326 nm, and the column temperature was 30°C. RESULTS: All the 7 compounds showed good linearity in the ranges of the test concentrations. The RSDs of the precision, stability and reproducibility tests were less than 3%. The average recoveries were in the range of 97.98%-99.29%. CONCLUSION: This method is simple, sensitive, accurate, and can be used for quality control of honeysuckle extract. Copyright 2012 by the Chinese Pharmaceutical Association.